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Comparison of E. coli O157:H7 preparation methods used for detection with surface plasmon resonance sensor
Sensors and Actuators B 107 (2005) 202–208
Comparison of E. coli O157:H7 preparation methods used for detection with surface plasmon resonance sensor Allen D. Taylora , Qiuming Yua , Shengfu Chena , Jiˇr´ı Homolaa,b , Shaoyi Jianga,∗ b
a Department of Chemical Engineering, University of Washington, Box 351750, Seattle, WA 98195, USA Institute of Radio Engineering and Electronics, Academy of Sciences, Chaberska 57, 18251 Prague, Czech Republic
Received 17 May 2004; received in revised form 5 October 2004; accepted 22 November 2004 Available online 17 February 2005
Abstract This paper reports the detection of Escherichia coli (E. coli) O157:H7 with a surface plasmon resonance (SPR) sensor using three sample preparation methods: untreated (viable), heat-killed then soaked in 70% ethanol, and detergent-lysed. The SPR sensing surface consists of a monoclonal antibody immobilized onto a mixed –COOH and –OH terminated self-assembled monolayer (SAM) of alkanethiols on a gold surface. The limit of detection (LOD) for each method is determined by the minimum measurable shift in resonant wavelength corresponding to the specific binding of E. coli O157:H7 and the subsequent binding of an antibody for amplification. Detergent-lysed samples produce the lowest LOD at 104 cfu/ml, while the LOD is 105 cfu/ml for heat-killed samples and 106 cfu/ml for untreated samples, respectively. Possible reasons for different limits of detection are discussed. © 2005 Elsevier B.V. All rights reserved. Keywords: Escherichia coli O157:H7; Foodborne pathogens; Surface plasmon resonance (SPR) sensor; Immunosensors; Self-assembled monolayer (SAM)
1. Introduction The Council for Agricultural Science and Technology estimates that as many as 9000 deaths and between 6.5 and 83 million illnesses are caused annually by microbial foodborne diseases, in the United States [1]. To reduce the deaths and illnesses caused by foodborne pathogens, a fast, sensitive, and reliable detection method is needed to identify contaminated foods. However, bacterial pathogens are difficult to rapidly detect and quantify at low concentrations. The methods currently used to detect bacteria are time-consuming. One example is the conventional microbiological culture method, which has one or more enrichment cycles and usually requires up to 36 h to obtain results. Immunologically based biosensor technologies such as amperometric immunosensors [2], potentiometric immunosensors [3], piezoelectric biosensors [4], electrical impedance biosensors [5], and optical biosen∗
Corresponding author. Tel.: +1 206 616 6509; fax: +1 206 543 3778. E-mail address: [email protected] (S. Jiang).
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sors [6] have been applied to the detection of bacteria [7]. They have the potential to provide rapid, sensitive, and specific detections. In this study, we used an optical sensor based on surface plasmon resonance (SPR) capable of real-time, quantitative, and label-free detections for the direct and continuous monitoring of bioanalytes. SPR sensors have been used to detect relatively low concentrations of proteins [8] and small molecules [9], but the limit of detection (LOD) for bacteria needs improvement. SPR detection of Escherichia coli (E. coli) O157:H7, Salmonella enteritidis (S. enteritidis), and Listeria monocytogenes (L. monocytogenes) have been previously reported in the literature [10,11]. Viable (untreated) E. coli O157:H7 had a LOD of 5 × 107 cfu/ml using a sandwich assay [10]. Heatkilled L. monocytogenes had a LOD of 106 cfu/ml for direct detection [11]. Heat-killed then ethanol soaked S. enteritidis had a LOD of 106 cfu/ml for direct detection [11]. In these cases, results are not directly comparable because different bacteria were used and samples were prepared by different methods. It is possible that the heat-killed L. monocytogenes
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and S. entritidis have a lower LOD than the viable E. coli O157:H7 due to the different methods used for sample preparation. However, there is a lack of systematic study that shows what method of sample preparation provides the lowest LOD using SPR or other immunosensors. Furthermore, the LOD for bacterial pathogens need to be improved beyond what was previously reported [10,11] since bacterial pathogens linked to foodborne illnesses have infectious doses ranging from 10 to 1000 cells [12]. This study investigates how the treatment of samples containing E. coli O157:H7 affects the detection of the bacteria with a SPR sensor. Three methods of preparing E. coli O157:H7 samples are compared: untreated (viable), heat-killed then ethanol soaked, and detergent-lysed.
2. Materials and methods 2.1. SPR instrumentation In this work, we used a SPR sensor developed at the Institute of Radio Engineering and Electronics (Prague, Czech Republic) [13,14] which is based on the Kretschman geometry of the attenuated total reflection (ATR) method and wavelength modulation (Fig. 1a). A BK7 glass chip coated with a gold SPR-active film is optically matched to a prism with immersion oil. An acrylic flow cell with a laser cut Mylar gasket is mechanically pressed to the chip and prism. A collimated polychromatic light beam is directed through the prism to the gold-coated substrate and excites surface plasmons at the metal–dielectric interface. The wavelength of light absorbed by the SPR is directly related to the refractive index of the dielectric film in contact with the thin metal surface (Fig. 1b). A sensorgram (Fig. 1c) records the shifts of res-
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onant wavelength as a function of time, which can be used to quantify the mass of captured analyte. The SPR sensor is equipped with two channels. One is the detection channel while the other is the reference channel. To compensate the detection channel for variations in temperature, bulk sample composition, and non-specific adsorption, the signal from the reference channel is subtracted from that of the detection channel. This is particularly important when determining the LOD. 2.2. Materials 2.2.1. Reagents 11-mercapto-1-undecanol (HSCH2 (CH2 )9 CH2 OH), 16mercaptohexadecanoic acid (HSCH2 (CH2 )13 CH2 COOH), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO). Bacterial protein extraction reagent (BPER) II was purchased from Pierce Biotechnology Inc. (Rockford, IL). Phosphate buffered saline (PBS) (0.01 M phosphate, 0.138 M sodium chloride, 0.0027 M potassium chloride, pH 7.4) was purchased from Sigma-Aldrich (St. Louis, MO). 2.2.2. Bacteria Stock cultures of E. coli O157:H7 (ATCC 700728) and Salmonella choleraesuis (S. choleraesuis) serotype typhimurium (ATCC 700720) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). E. coli K12 was a gift from Professor F. Baneyx at the University of Washington. Heat-killed L. monocytogenes was purchased from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD). 2.2.3. Antibodies Goat anti-E. coli O157:H7 polyclonal antibody (PAb) and Mouse anti-E. coli O157:H7 monoclonal antibody (MAb) were purchased from Biodesign International (Saco, ME). The MAb binds to the E. coli O157:H7 lipopolysacchride (LPS) and its O polysaccharide moiety. 2.3. Surface functionalization
Fig. 1. (a) SPR sensor based on the Kretschman geometry of the attenuated total reflection (ATR) method and wavelength modulation. A collimated polychromatic light beam is directed through the prism at a fixed angle and excites surface plasmon waves at the metal-dielectric interface. Binding of analyte in the dielectric to antibodies immobilized on the thin gold film causes changes in surface refractive index. (b) Reflectivity as a function of wavelength for two different refractive indices of the dielectric. The wavelength of light absorbed by the SPR, which is represented by the dip in the normalized reflected light recorded by the spectrophotometer, is quantitatively related to the refractive index of the dielectric film in contact with the thin gold film. (c) A sensorgram, which is a recording of resonant wavelength shift versus time. This idealized sensorgram shows the association due to binding of analyte on the surface, followed by dissociation of loosely bound analyte, then association due to binding of amplification antibody followed by dissociation of loosely bound antibody.
Glass chips are coated with a 2 nm adhesion promoting chromium film and then a 50 nm surface plasmon active gold film, both deposited by e-beam evaporation in vacuum. The gold surface is cleaned before the subsequent formation of a mixed SAM by washing with absolute ethanol, drying with nitrogen, stripping organic contaminants in an UV ozone cleaner for 20 min, then washing the surface with 18.2 M cm DI water and absolute ethanol, and drying with nitrogen. The surface is subsequently functionalized by the formation of a mixed SAM by incubating the cleaned substrate in ethanol with 0.7 mM 11-mercapto-1undecanol (C11 OH) and 0.3 mM 16-mercaptohexadecanoic acid (C15 COOH) at room temperature for approximately
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24 h. After the formation of the mixed SAM, the substrate is removed from the solution and washed with 10% acetic acid in ethanol and absolute ethanol to remove any alkanethiols not bound directly to the gold surface [8,15,16]. The chain lengths and the ratio of the mixed alkanethiol SAM were chosen, based on previous studies [15,16], for optimum protein detection. 2.4. Antibody immobilization The anti-E. coli O157:H7 MAb is immobilized onto the surface of the mixed SAM by reacting amines on the MAb to the carboxyl terminal group of the mixed SAM using EDC/NHS coupling chemistry. The procedure for MAb immobilization is as follows [8]. The carboxyl terminal group of the mixed SAM is activated by incubating the substrate in a stirring solution of 1.33 mg/ml NHS and 1.33 mg/ml EDC in dioxane/water (14:1) mixture for 1 h at room temperature. The substrate is then removed from the solution, rinsed with 18.2 M cm DI water, and dried with nitrogen. The MAb is linked to the activated surface by putting a 10 l drop of 1 mg/ml anti-E. coli O157:H7 MAb in PBS onto the surface, covering the surface with a glass cover slip, and then incubating the MAb with the activated surface for approximately 24 h at 4 ◦ C in a humidity-controlled petri dish. The antibodyfunctionalized substrate is washed with 18.2 M cm DI water and dried with nitrogen before SPR experiments. Since many amine groups are available on the MAb for reaction, the orientation of the MAb on the surface is random. Therefore, not every antibody is in a desirable orientation capable of binding antigens. The MAb is used throughout this study as the immobilized antibody. 2.5. Sample preparation for bacteria Stocks of bacteria are stored frozen at −80 ◦ C in LuriaBertani (LB) broth containing 16% glycerol. The bacteria are resuscitated by inoculating 5 ml of sterile LB broth in a test tube from the frozen stock of bacteria and incubating 16–20 h at 37 ◦ C in a reciprocal shaking water bath. The overnight culture of bacteria is enumerated using 10-fold serial dilutions plated on LB agar plates incubated at 37 ◦ C. For all treatment methods, 1.5 ml samples of overnight E. coli cultures were pelletized in a centrifuge at 10,000 × g for 10 min and the supernatant was discarded. Untreated samples were then resuspended in 1.5 ml of PBS, pelletized again at 10,000 × g for 10 min, and resuspended in 1.5 ml of PBS with 1 mg/ml BSA (PBS/BSA). Heat-killed then soaked in ethanol samples were resuspended in 450 l of PBS, heated to 90 ◦ C for one hour, diluted to 70% ethanol by adding 1050 l of ethanol, soaked for 30 min, then pelletized again at 10,000 × g for 10 min, and resuspended in 1.5 ml of PBS/BSA [11]. Detergentlysed samples were resuspended in 1.5 ml of PBS, pelletized again at 10,000 × g for 10 min, resuspended in 150 l of BPER II reagent for 10 min, and then diluted
to 1.5 ml by adding 1350 l of PBS/BSA. Serial dilution by a factor of 10 was performed for each sample in PBS/BSA. 2.6. SPR detection of E. coli O157:H7 PBS is flowed across the antibody-immobilized sensor surface to establish a baseline. PBS/BSA is then flowed through the SPR system to passivate all surfaces to minimize non-specific adsorption. Then, samples containing diluted E. coli O157:H7 in PBS/BSA are flowed for 20 min for direct detection and subsequently, the surface is washed with PBS/BSA for 10 min. A sandwich assay was performed to amplify the direct response for concentrations having less than 1 nm of resonant wavelength shift for the direct detection, by flowing 50 g/ml of MAb in PBS/BSA for 20 min followed by a 10 min wash with PBS/BSA. A substrate with a fresh SPR active gold film, SAM, and MAbs is used for each measurement. All SPR experiments are performed at a flow rate of 50 l/min.
3. Results and discussion 3.1. Detection of E. coli O157:H7 prepared by three methods A SPR sensor was used to detect samples containing E. coli O157:H7, which are prepared by three methods: untreated (viable), heat-killed then ethanol soaked, and detergent-lysed. Figs. 2–4 show the reference-compensated sensorgrams for each of the treatment methods at various concentrations. The reference-compensated sensorgrams are obtained by subtracting the resonant wavelength in the reference channel from that of the detection channel to com-
Fig. 2. Reference-compensated sensorgram of the direct detection and amplification of untreated E. coli O157:H7 samples at various concentrations (cfu/ml). The steps recorded by the sensorgrams are: 0–10 min—PBS with 1 mg/ml BSA, 10–30 min—direct detection response to prepared E. coli O157:H7 sample, 30–40 min—PBS with 1 mg/ml BSA, 40–60 min—amplification response to 50 g/ml MAb, and 60–70 min—PBS with 1 mg/ml BSA.
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Fig. 3. Reference-compensated sensorgram of the direct detection and amplification of heat-killed then soaked in ethanol E. coli O157:H7 samples at various concentrations (cfu/ml). The steps recorded by the sensorgrams are: 0–10 min—PBS with 1 mg/ml BSA, 10–30 min—direct detection response to prepared E. coli O157:H7 sample, 30–40 min—PBS with 1 mg/ml BSA, 40–50 min—amplification response to 50 g/ml MAb, and 50–60 min—PBS with 1 mg/ml BSA.
pensate variations in temperature, sample composition, and non-specific adsorption. Untreated (viable) E. coli O157:H7. The direct detection of untreated (viable) cells at concentrations of 109 and 108 cfu/ml produced reference-compensated resonant wavelength shifts of 7.3 and 2 nm, respectively, as shown in Fig. 2. The SPR response for the direct detection of untreated cells at a concentration of 107 cfu/ml was less than 1 nm. Because of the low direct detection response, a sandwich assay was used to detect untreated cells with concentrations less than or equal to107 cfu/ml by flowing in 50 ug/ml MAb for 20 min. Fig. 2 shows that the amplified detection of untreated cells at concentrations of 107 and 106 cfu/ml produced reference-compensated resonant wavelength shifts of 0.51 and 0.1 nm, respectively. The LOD for untreated sample with
Fig. 4. Reference-compensated sensorgram showing of the direct detection and amplification of detergent-lysed E. coli O157:H7 samples at various concentrations (cfu/ml). The steps recorded by the sensorgrams are: 0–10 min—PBS with 1 mg/ml BSA, 10–30 min—direct detection response to prepared E. coli O157:H7 sample, 30–40 min—PBS with 1 mg/ml BSA, 40–60 min—amplification response to 50 g/ml MAb, and 60–70 min—PBS with 1 mg/ml BSA.
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antibody amplification is 106 cfu/ml. Fratamico et al. [10] previously reported the SPR detection of untreated (viable) E. coli O157:H7. In their study, a LOD of 5–7 × 107 cfu/ml was achieved with an amplification antibody [10]. The different limits of detection from the two studies may be attributed to the use of different antibodies, surface chemistries, and SPR sensors. Heat-killed then soaked in ethanol E. coli O157:H7. The direct detection of E. coli O157:H7 samples that were heatkilled and ethanol soaked at concentrations of 109 , 108 and 107 cfu/ml have reference-compensated resonant wavelength shifts of 6.99, 4.93, and 1.82 nm, respectively, as shown in Fig. 3. Samples at concentrations less than or equal to106 cfu/ml were detected using direct direction and MAb amplification. Fig. 3 shows that the amplified detection of 106 and 105 cfu/ml yielded reference-compensated responses of 2.5 and 0.51 nm, respectively. E. coli O157:H7 samples were also prepared by heat-killing only. Results from both heatkilled then soaked in ethanol and heat-killed only are similar (data not shown). The sample preparation methods used in this study are similar to those used by Koubova et al., by which heat-killed L. monocytogenes and heat-killed then soaked in ethanol S. enteritidis are prepared. The LOD for E. coli O157:H7 using direct detection in this study is 106 cfu/ml for both heat-killed and heat-killed then soaked in ethanol, which is the same LOD reported by Koubova et al. for different bacteria with the same treatment methods. In this study, a lower LOD of 105 cfu/ml is achieved by secondary antibody (MAb) for amplification. For the sample preparation using the heat-killed and soaked in ethanol in Fig. 3, the sensorgrams for the concentrations of 108 and 109 cfu/ml have a more pronounced reflective index change when the sample is switched to the PBS/BSA due to residual ethanol in the sample from the sample preparation. Detergent-lysed E. coli O157:H7. In order to improve the LOD for bacteria using a SPR sensor, we treated the samples with a detergent to lyse the bacteria. Detergentlysing is a common method used in microbiology to obtain materials encapsulated inside a bacterium. However, this method has not been previously used to prepare samples of bacteria for SPR detection. As shown in Fig. 4, the direct detection of detergent-lysed samples at concentrations of 109 –106 cfu/ml yielded reference-compensated resonant wavelength shifts of 13.75, 6.18, 5.28, and 1.7 nm, respectively. The amplified detection of detergent-lysed samples at 105 and 104 cfu/ml yielded reference-compensated responses of 5.72 and 0.29 ± 0.14 nm, respectively. The lowest detection of 104 cfu/ml was reproduced in four separate experiments and the compensated SPR response reported is the average of the four experiments. Comparison of three sample preparation methods. Fig. 5a summarizes the resonant wavelength shifts for the direct detection of E. coli O157:H7 for each of the treatment methods. The LOD for the direct detection of detergent-lysed samples is 105 cfu/ml, while the LOD is 106 and 107 cfu/ml for the direct detection of heat-killed and untreated samples, respec-
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Fig. 5. Resonant wavelength shift versus concentration of bacteria for the detection of E. coli O157:H7 comparing untreated, heat-killed then ethanol soaked, and detergent-lysed samples by (a) direct detection and (b) amplification of direct detection by the subsequent exposure to a MAb reactive with E. coli O157:H7 (sandwich assay).
tively. Amplification with the MAb improved the LOD for each of the treatment methods by one order of magnitude. As shown in Fig. 5b, amplified detergent-lysed samples produce the lowest LOD at 104 cfu/ml, while the LOD is 105 and 106 cfu/ml for amplified heat-killed and untreated samples, respectively. The ability of large analytes, like bacteria, to bind to the antibody immobilized on the sensing surface is limited by diffusion and hydrodynamic effects. Each bacterium surface contains many antigens. Ideally, it is desirable to break the bacterium into small pieces of uniform size, which will not only increase the number of analytes, but also improve the diffusion of the analytes. Our experiments demonstrate that the sample preparation methods affect the LOD of E. coli O157:H7 using a SPR sensor due to changing the size of analytes. Heat-killed E. coli O157:H7 cells could either become spherical from their rod shape when they are killed or be broken, improving the LOD as compared to untreated sample. Detergent-lysing breaks the cells into small pieces, changing the size of the analytes and detectable antigen concentration dramatically. Therefore, detergent-lysed samples yield the lowest LOD among the three sample preparation methods. Further investigation of the size distribution of the analytes from each treatment method will provide more information regarding how sample treatment affects the LOD for SPR detection. In our current experiments, the flow conditions were fixed. As mentioned earlier, hydrodynamics plays a significant role in the sensing of E. coli samples. Since the SPR detection of bacteria is largely limited by the ability of the analytes to reach and bind to the surface, the flow-conditions can be optimized to improve the LOD of E. coli samples. 3.2. Non-fouling properties of sensing surfaces and the specificity of the antibody In SPR experiments the net resonant wavelength shift corresponds to the binding of E. coli O157:H7 to the immobilized antibody on the sensing surface and the subsequent binding of the secondary antibody to E. coli O157:H7 for amplification.
Therefore, it is necessary to test the resistance of the sensing surface to the nonspecific binding of bacteria and proteins and the specificity of the antibody. The fouling and the specificity of the antibody-immobilized sensing surface to bacteria were tested using three non-specific bacteria, E. coli K12 serotype, S. choleraesuis, and L. monocytogenes. Generally, it is difficult to produce surfaces that are non-fouling to bacteria [17]. The anti-E. coli O157:H7 MAb immobilized on the sensing surface and used for amplification binds specifically to E. coli O157:H7 LPS and its O polysaccharide moiety. Thus, it should not bind with E. coli K12 or other bacteria. Fig. 6 shows the SPR response to the binding of detergent-lysed E. coli K12 samples at various concentrations to the anti-E. coli O157:H7 MAb immobilized surface. Negligible sensor response was observed for detergent-lysed E. coli K12 at concentrations less than or equal to 106 cfu/ml. At 107 and 108 cfu/ml the sensor response was 0.1 and 0.3 nm, respectively. Secondary antibody (MAb) was used to probe the sur-
Fig. 6. Control experiments demonstrating that the surface is relatively nonfouling to E. coli K12 serotype and that the MAb does not react with E. coli K12 serotype. Detergent-lysed samples of E. coli K12 are flowed over the surface at 10 min intervals, each followed by 10 min of PBS. Each sample of detergent-lysed E. coli K12 is increased in concentration by 10-fold from 104 to 108 cfu/ml. Subsequently 50 ug/ml of MAb in PBS is flowed over the sensing surface.
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face exposed to detergent-lysed E. coli K12 at concentration of 108 cfu/ml and no net response was observed. This further indicates that the MAb does not bind to the E. coli K12 serotype. The 0.3 nm wavelength shift may be caused by the non-specific binding of detergent-lysed E. coli K12 samples onto the sensing surface. In addition, the sensing surface was also tested for non-specific binding against detergent-lysed samples containing 108 cfu/ml S. choleraesuis or 107 cfu/ml L. monocytogenes. A net resonant wavelength shift of 0.1 and 0.45 nm was observed, respectively. Subsequent exposure of these two surfaces to secondary antibody (MAb) showed no net response, further proving the specificity of the MAb. It should be pointed out that the non-specific responses associated with the E. coli K12, S. choleraesuis, and L. monocytogenes occur only at high concentrations of non-target bacteria and are significantly less than the specific response to E. coli O157:H7 at corresponding concentrations. The resistance of the sensing surface to non-specific protein adsorption was also tested. It was shown previously that the SAM-based platform used in these experiments was relatively non-fouling [8]. With 1 mg/ml BSA in PBS control experiments demonstrated no non-specific binding on the surface. For those experiments with MAb for amplification, the response of the MAb in the reference channel could be up to 0.3 nm in some cases. In all SPR experiments, the net responses are reported, which subtract the non-specific binding from the response in the detection channel.
4. Conclusions This study shows that the method used to prepare samples of bacteria for SPR detections has a significant effect on the limit of detection. When E. coli O157:H7 samples were treated with a detergent, the LOD was decreased by two orders of magnitude as compared to untreated samples and one order of magnitude as compared to heat-killed samples due to the change in the size of the analytes. The anti-E. coli O157:H7 monoclonal antibody proved to be specific. Using the MAb for amplification can not only improve LOD, but also avoid false alarms. The results suggest that the binding of E. coli O157:H7 to the antibody-immobilized substrate is diffusion-limited. Therefore, the LOD can be further improved by optimizing flow-conditions and sample treatment methods.
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Acknowledgements This project is funded by a grant from the U.S. Food and Drug Administration (FD-U-002250). Allen D. Taylor was partially supported by the Graduate Opportunities and Minority Achievement Award Fellowship at the University of Washington. We thank Professor Franc¸ois Baneyx at the University of Washington for providing E. coli K12 serotype.
Biographies Allen D. Taylor received his BS degree from the Department of Chemical Engineering at Texas A&M University in 2000. He is currently a PhD candidate in the Department of Chemical Engineering at the University of Washington, Seattle. His research interests are biosensors and surfaces relating to biomedical applications and tissue engineering.
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Qiuming Yu received her PhD degree from the Department of Chemical Engineering at Cornell University in 1995. She was a postdoctoral fellow at the Jet Propulsion Laboratory/California Institute of Technology between 1995 and 1996 and was an assistant professor in the Department of Chemical Engineering at Kansas State University between 1997 and 1999. She is currently a research scientist in the Departments of Physics and Chemical Engineering at the University of Washington, Seattle. Her research focuses on surface science and engineering.
Engineering and Electronics, Prague (Czech Republic). He also is affiliate associate professor at the University of Washington, Seattle (USA). His research interests are in photonics and biophotonics with emphasis on optical sensors and biosensors. He has contributed to two books and authored over 40 research papers in scientific journals and 80 conference contributions. J. Homola is a member of editorial boards of Sensors and Actuators B and Biosensors and Bioelectronics and senior member of IEEE.
Shengfu Chen received his PhD degree from Shanghai Institute of Nuclear Research, Chinese Academy of Sciences, China, in 1998. He has been a research associate at the University of Washington since 2000. His current research focuses on controlling nano-scale surface properties for protein adsorption, particularly protein orientation and surfaces to resist protein adsorption for applications in biosensors and biomaterials.
Shaoyi Jiang received his PhD degree from the Department of Chemical Engineering at Cornell University in 1993. He was a postdoctoral fellow in the Department of Chemistry at the University of California, Berkeley and a research fellow in the Department of Chemistry at California Institute of Technology. Currently, he is an associate professor in the Department of Chemical Engineering at the University of Washington, Seattle. His research focuses on molecular engineering of biological interfaces for biosensors and biomaterials.
Jiˇr´ı Homola (MS 1988, PhD 1993) is head of Photonics Division and chairman of Department of Optical Sensors at the Institute of Radio
Comparison of E. coli O157:H7 preparation methods used for detection with surface plasmon resonance sensor Allen D. Taylora , Qiuming Yua , Shengfu Chena , Jiˇr´ı Homolaa,b , Shaoyi Jianga,∗ b
a Department of Chemical Engineering, University of Washington, Box 351750, Seattle, WA 98195, USA Institute of Radio Engineering and Electronics, Academy of Sciences, Chaberska 57, 18251 Prague, Czech Republic
Received 17 May 2004; received in revised form 5 October 2004; accepted 22 November 2004 Available online 17 February 2005
Abstract This paper reports the detection of Escherichia coli (E. coli) O157:H7 with a surface plasmon resonance (SPR) sensor using three sample preparation methods: untreated (viable), heat-killed then soaked in 70% ethanol, and detergent-lysed. The SPR sensing surface consists of a monoclonal antibody immobilized onto a mixed –COOH and –OH terminated self-assembled monolayer (SAM) of alkanethiols on a gold surface. The limit of detection (LOD) for each method is determined by the minimum measurable shift in resonant wavelength corresponding to the specific binding of E. coli O157:H7 and the subsequent binding of an antibody for amplification. Detergent-lysed samples produce the lowest LOD at 104 cfu/ml, while the LOD is 105 cfu/ml for heat-killed samples and 106 cfu/ml for untreated samples, respectively. Possible reasons for different limits of detection are discussed. © 2005 Elsevier B.V. All rights reserved. Keywords: Escherichia coli O157:H7; Foodborne pathogens; Surface plasmon resonance (SPR) sensor; Immunosensors; Self-assembled monolayer (SAM)
1. Introduction The Council for Agricultural Science and Technology estimates that as many as 9000 deaths and between 6.5 and 83 million illnesses are caused annually by microbial foodborne diseases, in the United States [1]. To reduce the deaths and illnesses caused by foodborne pathogens, a fast, sensitive, and reliable detection method is needed to identify contaminated foods. However, bacterial pathogens are difficult to rapidly detect and quantify at low concentrations. The methods currently used to detect bacteria are time-consuming. One example is the conventional microbiological culture method, which has one or more enrichment cycles and usually requires up to 36 h to obtain results. Immunologically based biosensor technologies such as amperometric immunosensors [2], potentiometric immunosensors [3], piezoelectric biosensors [4], electrical impedance biosensors [5], and optical biosen∗
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sors [6] have been applied to the detection of bacteria [7]. They have the potential to provide rapid, sensitive, and specific detections. In this study, we used an optical sensor based on surface plasmon resonance (SPR) capable of real-time, quantitative, and label-free detections for the direct and continuous monitoring of bioanalytes. SPR sensors have been used to detect relatively low concentrations of proteins [8] and small molecules [9], but the limit of detection (LOD) for bacteria needs improvement. SPR detection of Escherichia coli (E. coli) O157:H7, Salmonella enteritidis (S. enteritidis), and Listeria monocytogenes (L. monocytogenes) have been previously reported in the literature [10,11]. Viable (untreated) E. coli O157:H7 had a LOD of 5 × 107 cfu/ml using a sandwich assay [10]. Heatkilled L. monocytogenes had a LOD of 106 cfu/ml for direct detection [11]. Heat-killed then ethanol soaked S. enteritidis had a LOD of 106 cfu/ml for direct detection [11]. In these cases, results are not directly comparable because different bacteria were used and samples were prepared by different methods. It is possible that the heat-killed L. monocytogenes
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and S. entritidis have a lower LOD than the viable E. coli O157:H7 due to the different methods used for sample preparation. However, there is a lack of systematic study that shows what method of sample preparation provides the lowest LOD using SPR or other immunosensors. Furthermore, the LOD for bacterial pathogens need to be improved beyond what was previously reported [10,11] since bacterial pathogens linked to foodborne illnesses have infectious doses ranging from 10 to 1000 cells [12]. This study investigates how the treatment of samples containing E. coli O157:H7 affects the detection of the bacteria with a SPR sensor. Three methods of preparing E. coli O157:H7 samples are compared: untreated (viable), heat-killed then ethanol soaked, and detergent-lysed.
2. Materials and methods 2.1. SPR instrumentation In this work, we used a SPR sensor developed at the Institute of Radio Engineering and Electronics (Prague, Czech Republic) [13,14] which is based on the Kretschman geometry of the attenuated total reflection (ATR) method and wavelength modulation (Fig. 1a). A BK7 glass chip coated with a gold SPR-active film is optically matched to a prism with immersion oil. An acrylic flow cell with a laser cut Mylar gasket is mechanically pressed to the chip and prism. A collimated polychromatic light beam is directed through the prism to the gold-coated substrate and excites surface plasmons at the metal–dielectric interface. The wavelength of light absorbed by the SPR is directly related to the refractive index of the dielectric film in contact with the thin metal surface (Fig. 1b). A sensorgram (Fig. 1c) records the shifts of res-
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onant wavelength as a function of time, which can be used to quantify the mass of captured analyte. The SPR sensor is equipped with two channels. One is the detection channel while the other is the reference channel. To compensate the detection channel for variations in temperature, bulk sample composition, and non-specific adsorption, the signal from the reference channel is subtracted from that of the detection channel. This is particularly important when determining the LOD. 2.2. Materials 2.2.1. Reagents 11-mercapto-1-undecanol (HSCH2 (CH2 )9 CH2 OH), 16mercaptohexadecanoic acid (HSCH2 (CH2 )13 CH2 COOH), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO). Bacterial protein extraction reagent (BPER) II was purchased from Pierce Biotechnology Inc. (Rockford, IL). Phosphate buffered saline (PBS) (0.01 M phosphate, 0.138 M sodium chloride, 0.0027 M potassium chloride, pH 7.4) was purchased from Sigma-Aldrich (St. Louis, MO). 2.2.2. Bacteria Stock cultures of E. coli O157:H7 (ATCC 700728) and Salmonella choleraesuis (S. choleraesuis) serotype typhimurium (ATCC 700720) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). E. coli K12 was a gift from Professor F. Baneyx at the University of Washington. Heat-killed L. monocytogenes was purchased from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD). 2.2.3. Antibodies Goat anti-E. coli O157:H7 polyclonal antibody (PAb) and Mouse anti-E. coli O157:H7 monoclonal antibody (MAb) were purchased from Biodesign International (Saco, ME). The MAb binds to the E. coli O157:H7 lipopolysacchride (LPS) and its O polysaccharide moiety. 2.3. Surface functionalization
Fig. 1. (a) SPR sensor based on the Kretschman geometry of the attenuated total reflection (ATR) method and wavelength modulation. A collimated polychromatic light beam is directed through the prism at a fixed angle and excites surface plasmon waves at the metal-dielectric interface. Binding of analyte in the dielectric to antibodies immobilized on the thin gold film causes changes in surface refractive index. (b) Reflectivity as a function of wavelength for two different refractive indices of the dielectric. The wavelength of light absorbed by the SPR, which is represented by the dip in the normalized reflected light recorded by the spectrophotometer, is quantitatively related to the refractive index of the dielectric film in contact with the thin gold film. (c) A sensorgram, which is a recording of resonant wavelength shift versus time. This idealized sensorgram shows the association due to binding of analyte on the surface, followed by dissociation of loosely bound analyte, then association due to binding of amplification antibody followed by dissociation of loosely bound antibody.
Glass chips are coated with a 2 nm adhesion promoting chromium film and then a 50 nm surface plasmon active gold film, both deposited by e-beam evaporation in vacuum. The gold surface is cleaned before the subsequent formation of a mixed SAM by washing with absolute ethanol, drying with nitrogen, stripping organic contaminants in an UV ozone cleaner for 20 min, then washing the surface with 18.2 M cm DI water and absolute ethanol, and drying with nitrogen. The surface is subsequently functionalized by the formation of a mixed SAM by incubating the cleaned substrate in ethanol with 0.7 mM 11-mercapto-1undecanol (C11 OH) and 0.3 mM 16-mercaptohexadecanoic acid (C15 COOH) at room temperature for approximately
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24 h. After the formation of the mixed SAM, the substrate is removed from the solution and washed with 10% acetic acid in ethanol and absolute ethanol to remove any alkanethiols not bound directly to the gold surface [8,15,16]. The chain lengths and the ratio of the mixed alkanethiol SAM were chosen, based on previous studies [15,16], for optimum protein detection. 2.4. Antibody immobilization The anti-E. coli O157:H7 MAb is immobilized onto the surface of the mixed SAM by reacting amines on the MAb to the carboxyl terminal group of the mixed SAM using EDC/NHS coupling chemistry. The procedure for MAb immobilization is as follows [8]. The carboxyl terminal group of the mixed SAM is activated by incubating the substrate in a stirring solution of 1.33 mg/ml NHS and 1.33 mg/ml EDC in dioxane/water (14:1) mixture for 1 h at room temperature. The substrate is then removed from the solution, rinsed with 18.2 M cm DI water, and dried with nitrogen. The MAb is linked to the activated surface by putting a 10 l drop of 1 mg/ml anti-E. coli O157:H7 MAb in PBS onto the surface, covering the surface with a glass cover slip, and then incubating the MAb with the activated surface for approximately 24 h at 4 ◦ C in a humidity-controlled petri dish. The antibodyfunctionalized substrate is washed with 18.2 M cm DI water and dried with nitrogen before SPR experiments. Since many amine groups are available on the MAb for reaction, the orientation of the MAb on the surface is random. Therefore, not every antibody is in a desirable orientation capable of binding antigens. The MAb is used throughout this study as the immobilized antibody. 2.5. Sample preparation for bacteria Stocks of bacteria are stored frozen at −80 ◦ C in LuriaBertani (LB) broth containing 16% glycerol. The bacteria are resuscitated by inoculating 5 ml of sterile LB broth in a test tube from the frozen stock of bacteria and incubating 16–20 h at 37 ◦ C in a reciprocal shaking water bath. The overnight culture of bacteria is enumerated using 10-fold serial dilutions plated on LB agar plates incubated at 37 ◦ C. For all treatment methods, 1.5 ml samples of overnight E. coli cultures were pelletized in a centrifuge at 10,000 × g for 10 min and the supernatant was discarded. Untreated samples were then resuspended in 1.5 ml of PBS, pelletized again at 10,000 × g for 10 min, and resuspended in 1.5 ml of PBS with 1 mg/ml BSA (PBS/BSA). Heat-killed then soaked in ethanol samples were resuspended in 450 l of PBS, heated to 90 ◦ C for one hour, diluted to 70% ethanol by adding 1050 l of ethanol, soaked for 30 min, then pelletized again at 10,000 × g for 10 min, and resuspended in 1.5 ml of PBS/BSA [11]. Detergentlysed samples were resuspended in 1.5 ml of PBS, pelletized again at 10,000 × g for 10 min, resuspended in 150 l of BPER II reagent for 10 min, and then diluted
to 1.5 ml by adding 1350 l of PBS/BSA. Serial dilution by a factor of 10 was performed for each sample in PBS/BSA. 2.6. SPR detection of E. coli O157:H7 PBS is flowed across the antibody-immobilized sensor surface to establish a baseline. PBS/BSA is then flowed through the SPR system to passivate all surfaces to minimize non-specific adsorption. Then, samples containing diluted E. coli O157:H7 in PBS/BSA are flowed for 20 min for direct detection and subsequently, the surface is washed with PBS/BSA for 10 min. A sandwich assay was performed to amplify the direct response for concentrations having less than 1 nm of resonant wavelength shift for the direct detection, by flowing 50 g/ml of MAb in PBS/BSA for 20 min followed by a 10 min wash with PBS/BSA. A substrate with a fresh SPR active gold film, SAM, and MAbs is used for each measurement. All SPR experiments are performed at a flow rate of 50 l/min.
3. Results and discussion 3.1. Detection of E. coli O157:H7 prepared by three methods A SPR sensor was used to detect samples containing E. coli O157:H7, which are prepared by three methods: untreated (viable), heat-killed then ethanol soaked, and detergent-lysed. Figs. 2–4 show the reference-compensated sensorgrams for each of the treatment methods at various concentrations. The reference-compensated sensorgrams are obtained by subtracting the resonant wavelength in the reference channel from that of the detection channel to com-
Fig. 2. Reference-compensated sensorgram of the direct detection and amplification of untreated E. coli O157:H7 samples at various concentrations (cfu/ml). The steps recorded by the sensorgrams are: 0–10 min—PBS with 1 mg/ml BSA, 10–30 min—direct detection response to prepared E. coli O157:H7 sample, 30–40 min—PBS with 1 mg/ml BSA, 40–60 min—amplification response to 50 g/ml MAb, and 60–70 min—PBS with 1 mg/ml BSA.
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Fig. 3. Reference-compensated sensorgram of the direct detection and amplification of heat-killed then soaked in ethanol E. coli O157:H7 samples at various concentrations (cfu/ml). The steps recorded by the sensorgrams are: 0–10 min—PBS with 1 mg/ml BSA, 10–30 min—direct detection response to prepared E. coli O157:H7 sample, 30–40 min—PBS with 1 mg/ml BSA, 40–50 min—amplification response to 50 g/ml MAb, and 50–60 min—PBS with 1 mg/ml BSA.
pensate variations in temperature, sample composition, and non-specific adsorption. Untreated (viable) E. coli O157:H7. The direct detection of untreated (viable) cells at concentrations of 109 and 108 cfu/ml produced reference-compensated resonant wavelength shifts of 7.3 and 2 nm, respectively, as shown in Fig. 2. The SPR response for the direct detection of untreated cells at a concentration of 107 cfu/ml was less than 1 nm. Because of the low direct detection response, a sandwich assay was used to detect untreated cells with concentrations less than or equal to107 cfu/ml by flowing in 50 ug/ml MAb for 20 min. Fig. 2 shows that the amplified detection of untreated cells at concentrations of 107 and 106 cfu/ml produced reference-compensated resonant wavelength shifts of 0.51 and 0.1 nm, respectively. The LOD for untreated sample with
Fig. 4. Reference-compensated sensorgram showing of the direct detection and amplification of detergent-lysed E. coli O157:H7 samples at various concentrations (cfu/ml). The steps recorded by the sensorgrams are: 0–10 min—PBS with 1 mg/ml BSA, 10–30 min—direct detection response to prepared E. coli O157:H7 sample, 30–40 min—PBS with 1 mg/ml BSA, 40–60 min—amplification response to 50 g/ml MAb, and 60–70 min—PBS with 1 mg/ml BSA.
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antibody amplification is 106 cfu/ml. Fratamico et al. [10] previously reported the SPR detection of untreated (viable) E. coli O157:H7. In their study, a LOD of 5–7 × 107 cfu/ml was achieved with an amplification antibody [10]. The different limits of detection from the two studies may be attributed to the use of different antibodies, surface chemistries, and SPR sensors. Heat-killed then soaked in ethanol E. coli O157:H7. The direct detection of E. coli O157:H7 samples that were heatkilled and ethanol soaked at concentrations of 109 , 108 and 107 cfu/ml have reference-compensated resonant wavelength shifts of 6.99, 4.93, and 1.82 nm, respectively, as shown in Fig. 3. Samples at concentrations less than or equal to106 cfu/ml were detected using direct direction and MAb amplification. Fig. 3 shows that the amplified detection of 106 and 105 cfu/ml yielded reference-compensated responses of 2.5 and 0.51 nm, respectively. E. coli O157:H7 samples were also prepared by heat-killing only. Results from both heatkilled then soaked in ethanol and heat-killed only are similar (data not shown). The sample preparation methods used in this study are similar to those used by Koubova et al., by which heat-killed L. monocytogenes and heat-killed then soaked in ethanol S. enteritidis are prepared. The LOD for E. coli O157:H7 using direct detection in this study is 106 cfu/ml for both heat-killed and heat-killed then soaked in ethanol, which is the same LOD reported by Koubova et al. for different bacteria with the same treatment methods. In this study, a lower LOD of 105 cfu/ml is achieved by secondary antibody (MAb) for amplification. For the sample preparation using the heat-killed and soaked in ethanol in Fig. 3, the sensorgrams for the concentrations of 108 and 109 cfu/ml have a more pronounced reflective index change when the sample is switched to the PBS/BSA due to residual ethanol in the sample from the sample preparation. Detergent-lysed E. coli O157:H7. In order to improve the LOD for bacteria using a SPR sensor, we treated the samples with a detergent to lyse the bacteria. Detergentlysing is a common method used in microbiology to obtain materials encapsulated inside a bacterium. However, this method has not been previously used to prepare samples of bacteria for SPR detection. As shown in Fig. 4, the direct detection of detergent-lysed samples at concentrations of 109 –106 cfu/ml yielded reference-compensated resonant wavelength shifts of 13.75, 6.18, 5.28, and 1.7 nm, respectively. The amplified detection of detergent-lysed samples at 105 and 104 cfu/ml yielded reference-compensated responses of 5.72 and 0.29 ± 0.14 nm, respectively. The lowest detection of 104 cfu/ml was reproduced in four separate experiments and the compensated SPR response reported is the average of the four experiments. Comparison of three sample preparation methods. Fig. 5a summarizes the resonant wavelength shifts for the direct detection of E. coli O157:H7 for each of the treatment methods. The LOD for the direct detection of detergent-lysed samples is 105 cfu/ml, while the LOD is 106 and 107 cfu/ml for the direct detection of heat-killed and untreated samples, respec-
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Fig. 5. Resonant wavelength shift versus concentration of bacteria for the detection of E. coli O157:H7 comparing untreated, heat-killed then ethanol soaked, and detergent-lysed samples by (a) direct detection and (b) amplification of direct detection by the subsequent exposure to a MAb reactive with E. coli O157:H7 (sandwich assay).
tively. Amplification with the MAb improved the LOD for each of the treatment methods by one order of magnitude. As shown in Fig. 5b, amplified detergent-lysed samples produce the lowest LOD at 104 cfu/ml, while the LOD is 105 and 106 cfu/ml for amplified heat-killed and untreated samples, respectively. The ability of large analytes, like bacteria, to bind to the antibody immobilized on the sensing surface is limited by diffusion and hydrodynamic effects. Each bacterium surface contains many antigens. Ideally, it is desirable to break the bacterium into small pieces of uniform size, which will not only increase the number of analytes, but also improve the diffusion of the analytes. Our experiments demonstrate that the sample preparation methods affect the LOD of E. coli O157:H7 using a SPR sensor due to changing the size of analytes. Heat-killed E. coli O157:H7 cells could either become spherical from their rod shape when they are killed or be broken, improving the LOD as compared to untreated sample. Detergent-lysing breaks the cells into small pieces, changing the size of the analytes and detectable antigen concentration dramatically. Therefore, detergent-lysed samples yield the lowest LOD among the three sample preparation methods. Further investigation of the size distribution of the analytes from each treatment method will provide more information regarding how sample treatment affects the LOD for SPR detection. In our current experiments, the flow conditions were fixed. As mentioned earlier, hydrodynamics plays a significant role in the sensing of E. coli samples. Since the SPR detection of bacteria is largely limited by the ability of the analytes to reach and bind to the surface, the flow-conditions can be optimized to improve the LOD of E. coli samples. 3.2. Non-fouling properties of sensing surfaces and the specificity of the antibody In SPR experiments the net resonant wavelength shift corresponds to the binding of E. coli O157:H7 to the immobilized antibody on the sensing surface and the subsequent binding of the secondary antibody to E. coli O157:H7 for amplification.
Therefore, it is necessary to test the resistance of the sensing surface to the nonspecific binding of bacteria and proteins and the specificity of the antibody. The fouling and the specificity of the antibody-immobilized sensing surface to bacteria were tested using three non-specific bacteria, E. coli K12 serotype, S. choleraesuis, and L. monocytogenes. Generally, it is difficult to produce surfaces that are non-fouling to bacteria [17]. The anti-E. coli O157:H7 MAb immobilized on the sensing surface and used for amplification binds specifically to E. coli O157:H7 LPS and its O polysaccharide moiety. Thus, it should not bind with E. coli K12 or other bacteria. Fig. 6 shows the SPR response to the binding of detergent-lysed E. coli K12 samples at various concentrations to the anti-E. coli O157:H7 MAb immobilized surface. Negligible sensor response was observed for detergent-lysed E. coli K12 at concentrations less than or equal to 106 cfu/ml. At 107 and 108 cfu/ml the sensor response was 0.1 and 0.3 nm, respectively. Secondary antibody (MAb) was used to probe the sur-
Fig. 6. Control experiments demonstrating that the surface is relatively nonfouling to E. coli K12 serotype and that the MAb does not react with E. coli K12 serotype. Detergent-lysed samples of E. coli K12 are flowed over the surface at 10 min intervals, each followed by 10 min of PBS. Each sample of detergent-lysed E. coli K12 is increased in concentration by 10-fold from 104 to 108 cfu/ml. Subsequently 50 ug/ml of MAb in PBS is flowed over the sensing surface.
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face exposed to detergent-lysed E. coli K12 at concentration of 108 cfu/ml and no net response was observed. This further indicates that the MAb does not bind to the E. coli K12 serotype. The 0.3 nm wavelength shift may be caused by the non-specific binding of detergent-lysed E. coli K12 samples onto the sensing surface. In addition, the sensing surface was also tested for non-specific binding against detergent-lysed samples containing 108 cfu/ml S. choleraesuis or 107 cfu/ml L. monocytogenes. A net resonant wavelength shift of 0.1 and 0.45 nm was observed, respectively. Subsequent exposure of these two surfaces to secondary antibody (MAb) showed no net response, further proving the specificity of the MAb. It should be pointed out that the non-specific responses associated with the E. coli K12, S. choleraesuis, and L. monocytogenes occur only at high concentrations of non-target bacteria and are significantly less than the specific response to E. coli O157:H7 at corresponding concentrations. The resistance of the sensing surface to non-specific protein adsorption was also tested. It was shown previously that the SAM-based platform used in these experiments was relatively non-fouling [8]. With 1 mg/ml BSA in PBS control experiments demonstrated no non-specific binding on the surface. For those experiments with MAb for amplification, the response of the MAb in the reference channel could be up to 0.3 nm in some cases. In all SPR experiments, the net responses are reported, which subtract the non-specific binding from the response in the detection channel.
4. Conclusions This study shows that the method used to prepare samples of bacteria for SPR detections has a significant effect on the limit of detection. When E. coli O157:H7 samples were treated with a detergent, the LOD was decreased by two orders of magnitude as compared to untreated samples and one order of magnitude as compared to heat-killed samples due to the change in the size of the analytes. The anti-E. coli O157:H7 monoclonal antibody proved to be specific. Using the MAb for amplification can not only improve LOD, but also avoid false alarms. The results suggest that the binding of E. coli O157:H7 to the antibody-immobilized substrate is diffusion-limited. Therefore, the LOD can be further improved by optimizing flow-conditions and sample treatment methods.
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Acknowledgements This project is funded by a grant from the U.S. Food and Drug Administration (FD-U-002250). Allen D. Taylor was partially supported by the Graduate Opportunities and Minority Achievement Award Fellowship at the University of Washington. We thank Professor Franc¸ois Baneyx at the University of Washington for providing E. coli K12 serotype.
Biographies Allen D. Taylor received his BS degree from the Department of Chemical Engineering at Texas A&M University in 2000. He is currently a PhD candidate in the Department of Chemical Engineering at the University of Washington, Seattle. His research interests are biosensors and surfaces relating to biomedical applications and tissue engineering.
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Qiuming Yu received her PhD degree from the Department of Chemical Engineering at Cornell University in 1995. She was a postdoctoral fellow at the Jet Propulsion Laboratory/California Institute of Technology between 1995 and 1996 and was an assistant professor in the Department of Chemical Engineering at Kansas State University between 1997 and 1999. She is currently a research scientist in the Departments of Physics and Chemical Engineering at the University of Washington, Seattle. Her research focuses on surface science and engineering.
Engineering and Electronics, Prague (Czech Republic). He also is affiliate associate professor at the University of Washington, Seattle (USA). His research interests are in photonics and biophotonics with emphasis on optical sensors and biosensors. He has contributed to two books and authored over 40 research papers in scientific journals and 80 conference contributions. J. Homola is a member of editorial boards of Sensors and Actuators B and Biosensors and Bioelectronics and senior member of IEEE.
Shengfu Chen received his PhD degree from Shanghai Institute of Nuclear Research, Chinese Academy of Sciences, China, in 1998. He has been a research associate at the University of Washington since 2000. His current research focuses on controlling nano-scale surface properties for protein adsorption, particularly protein orientation and surfaces to resist protein adsorption for applications in biosensors and biomaterials.
Shaoyi Jiang received his PhD degree from the Department of Chemical Engineering at Cornell University in 1993. He was a postdoctoral fellow in the Department of Chemistry at the University of California, Berkeley and a research fellow in the Department of Chemistry at California Institute of Technology. Currently, he is an associate professor in the Department of Chemical Engineering at the University of Washington, Seattle. His research focuses on molecular engineering of biological interfaces for biosensors and biomaterials.
Jiˇr´ı Homola (MS 1988, PhD 1993) is head of Photonics Division and chairman of Department of Optical Sensors at the Institute of Radio